The invention relates to molecules, compositions and methods that can be used for the treatment and prevention of HSV infection. More particularly, the invention identifies epitopes of HSV proteins that can be used for the development of methods, molecules and compositions having the antigenic specificity of HSV-specific T cells, and in particular, of CD8+ T cells.
Cellular immune responses are required to limit the severity of recurrent HSV infection in humans. Initial genital HSV-2 infections are prolonged and severe, while recurrences are less severe and more frequently asymptomatic. Resolution of primary HSV-2 infection is associated with infiltration of antigen-specific T cells, including CD8+ cytotoxic T lymphocytes (CTLs). Serial lesion biopsy studies of recurrent HSV-2 infection in humans has shown a shift to CD8+ predominance as lesions mature and correlation of local CTL activity with virus clearance (Koelle, D M et al., J. Clin. Invest. 1998, 101:1500-1508; Cunningham, A L et al., J. Clin. Invest. 1985, 75:226-233). Thus, HSV antigens recognized by CD8+ CTL can be used for novel therapies and vaccines.
The complete DNA sequence of herpes simplex virus (HSV) is approximately 150 kb and encodes about 85 known genes, each of which encodes a protein in the range of 50-1000 amino acids in length. Unknown are the immunogenic epitopes within these proteins, each epitope approximately 9-12 amino acids in length, that are capable of eliciting an effective T cell immune response to viral infection.
There remains a need to identify specific epitopes capable of eliciting an effective immune response to HSV infection. Such information can lead to the identification of more effective immunogenic antigens useful for the prevention and treatment of HSV infection.
The invention provides HSV antigens, polypeptides comprising HSV antigens, polynucleotides encoding the polypeptides, vectors, and recombinant viruses containing the polynucleotides, antigen-presenting cells (APCs) presenting the polypeptides, immune cells directed against HSV, and pharmaceutical compositions. The pharmaceutical compositions can be used both prophylactically and therapeutically. The antigens of the invention are recognized by T cells recovered from herpetic lesions. The invention additionally provides methods, including methods for preventing and treating HSV infection, for killing HSV-infected cells, for inhibiting viral replication, for enhancing secretion of antiviral and/or immunomodulatory lymphokines, and for enhancing production of HSV-specific antibody. For preventing and treating HSV infection, for enhancing secretion of antiviral and/or immunomodulatory lymphokines, for enhancing production of HSV-specific antibody, and generally for stimulating and/or augmenting HSV-specific immunity, the method comprises administering to a subject a polypeptide, polynucleotide, recombinant virus, APC, immune cell or composition of the invention. The methods for killing HSV-infected cells and for inhibiting viral replication comprise contacting an HSV-infected cell with an immune cell of the invention. The immune cell of the invention is one that has been stimulated by an antigen of the invention or by an APC that presents an antigen of the invention. A method for producing such immune cells is also provided by the invention. The method comprises contacting an immune cell with an APC, preferably a dendritic cell, that has been modified to present an antigen of the invention. In a preferred embodiment, the immune cell is a T cell such as a CD4+ or CD8+ T cell.
In one embodiment, the invention provides a composition comprising an HSV polypeptide. The polypeptide comprises an ICP0 or UL47 protein or a fragment thereof. In one embodiment, the fragment comprises amino acids 92-101 of ICP0 or a substitutional variant thereof. In other embodiments, the fragment comprises amino acids 289-298, 548-557, 550-559, 551-559 and/or 551-561 of UL47 or a substitutional variant thereof. Also provided is an isolated polynucleotide that encodes a polypeptide of the invention, and a composition comprising the polynucleotide. The invention additionally provides a recombinant virus genetically modified to express a polynucleotide of the invention, and a composition comprising the recombinant virus. In preferred embodiments, the virus is a vaccinia virus, canary pox virus, HSV, lentivirus, retrovirus or adenovirus. A composition of the invention can be a pharmaceutical composition. The composition can optionally comprise a pharmaceutically acceptable carrier and/or an adjuvant.
The invention additionally provides a method of identifying an immunogenic epitope of an infectious organism, such as a virus, bacterium or parasite. Preferably, the infectious organism is a virus, such as HSV. In one embodiment, the method comprises preparing a collection of random fragments of the organismal genome. The fragments. can be prepared using any of a variety of standard methods, including, but not limited to, digestion with restriction enzymes and mechanical fragmentation, such as by controlled sonication (Mougneau E et al., Science 1995, 268:563-66). In a preferred embodiment, the organism is HSV-2 and the fragments of viral genome are prepared by digestion with Sau3A I. Examples of other restriction enzymes that can be used include, but are not limited to, Apa I, Sma I, and Alu I. The fragments of genomic DNA are then ligated into a vector, preferably by using a partial fill-in reaction. A preferred vector is a member of the pcDNA3.1(+) his series. The fragments are then expressed using conventional techniques. Preferably, the expression is performed using a Cos-7 transfection method (De Plaen E et al. In: Lefkowits I, ed. Immunology Methods Manual, v. 2. New York: Academic Press, 1997:691-718). The Cos-7 cells can be co-transfected with an appropriate HLA molecule capable of presenting the target antigen.
The ability of the expressed polypeptide to elicit a cellular immune response is then assayed. Ability to elicit a cellular immune response is indicative of the presence of an immunogenic epitope. Assays that can be used to detect ability to elicit a cellular immune response include, but are not limited to, cytotoxicity assays and lymphokine secretion assays. In one embodiment, the assay is an interferon-gamma assay.
In a preferred embodiment, the invention provides a method for identifying HSV epitopes immunogenic for CD8+ T cells. The method comprises obtaining CD8+ T cells from an HSV lesion, and assaying the obtained T cells to identify T cells having ability to recognize HSV-infected cells. The method further comprises obtaining and fragmenting a nucleic acid preparation from HSV, expressing one or more fragments of the obtained nucleic acid, and assaying the expressed fragments for antigenic reactivity with the identified HSV-specific T cells. An expressed fragment having reactivity with the HSV-specific T cells is identified as encoding an HSV epitope immunogenic for CD8+ T cells.
The above steps can be repeated with sub fragments of the genome fragments. The method can further comprise sequencing a fragment of the genome. In one embodiment, the assaying of T cells comprises performing a cytotoxicity assay or an interferon-gamma assay. The assaying can be performed with an immune cell derived from a subject that has been exposed to the infectious organism. In preferred embodiments, the cell is derived from a site of active infection, such as skin or cervix, or from blood of an infected subject.
The invention further provides immunogenic epitopes identified by the method of the invention, polypeptides comprising the epitopes, and polynucleotides encoding the polypeptides. Suitable infectious organisms include bacteria, parasites and viruses. Examples of viruses include DNA and RNA viruses, both double-stranded and single-stranded. The method of the invention provides a strategy for combating a variety of infectious organisms, including those that exhibit significant variability, as knowledge of the organism""s nucleic acid sequence is not required.